Sodium channel blockers

ABSTRACT

The present invention relates to sodium channel blockers. The present invention also includes a variety of methods of treatment using these inventive sodium channel blockers.

CONTINUING APPLICATION DATA

This application is a Continuation of prior U.S. application Ser. No.10/973,474, filed on Oct. 27, 2004 now U.S. Pat. No. 7,026,325, nowallowed, which is a Divisional of application Ser. No. 10/367,947, filedon Feb. 19, 2003, U.S. Pat. No. 6,903,105, issued Jun. 7, 2005.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to sodium channel blockers. The presentinvention also includes a variety of methods of treatment using theseinventive sodium channel blockers.

2. Description of the Background

The mucosal surfaces at the interface between the environment and thebody have evolved a number of “innate defense”, i.e., protectivemechanisms. A principal form of such innate defense is to cleanse thesesurfaces with liquid. Typically, the quantity of the liquid layer on amucosal surface reflects the balance between epithelial liquidsecretion, often reflecting anion (Cl⁻ and/or HCO₃ ⁻) secretion coupledwith water (and a cation counter-ion), and epithelial liquid absorption,often reflecting Na⁺ absorption, coupled with water and counter anion(Cl⁻ and/or HCO₃ ⁻). Many diseases of mucosal surfaces are caused by toolittle protective liquid on those mucosal surfaces created by animbalance between secretion (too little) and absorption (relatively toomuch). The defective salt transport processes that characterize thesemucosal dysfunctions reside in the epithelial layer of the mucosalsurface.

One approach to replenish the protective liquid layer on mucosalsurfaces is to “re-balance” the system by blocking Na⁺ channel andliquid absorption. The epithelial protein that mediates therate-limiting step of Na⁺ and liquid absorption is the epithelial Na⁺channel (ENaC). ENaC is positioned on the apical surface of theepithelium, i.e. the mucosal surface-environmental interface. Therefore,to inhibit ENaC mediated Na⁺ and liquid absorption, an ENaC blocker ofthe amiloride class (which blocks from the extracellular domain of ENaC)must be delivered to the mucosal surface and, importantly, be maintainedat this site, to achieve therapeutic utility. The present inventiondescribes diseases characterized by too little liquid on mucosalsurfaces and “topical” sodium channel blockers designed to exhibit theincreased potency, reduced mucosal absorption, and slow dissociation(“unbinding” or detachment) from ENaC required for therapy of thesediseases.

Chronic bronchitis (CB), including the most common lethal genetic formof chronic bronchitis, cystic fibrosis (CF), are diseases that reflectthe body's failure to clear mucus normally from the lungs, whichultimately produces chronic airways infection. In the normal lung, theprimary defense against chronic intrapulmonary airways infection(chronic bronchitis) is mediated by the continuous clearance of mucusfrom bronchial airway surfaces. This function in health effectivelyremoves from the lung potentially noxious toxins and pathogens. Recentdata indicate that the initiating problem, i.e., the “basic defect,” inboth CB and CF is the failure to clear mucus from airway surfaces. Thefailure to clear mucus reflects an imbalance between the amount ofliquid and mucin on airway surfaces. This “airway surface liquid” (ASL)is primarily composed of salt and water in proportions similar to plasma(i.e., isotonic). Mucin macromolecules organize into a well defined“mucus layer” which normally traps inhaled bacteria and is transportedout of the lung via the actions of cilia which beat in a watery, lowviscosity solution termed the “periciliary liquid” (PCL). In the diseasestate, there is an imbalance in the quantities of mucus as ASL on airwaysurfaces. This results in a relative reduction in ASL which leads tomucus concentration, reduction in the lubricant activity of the PCL, anda failure to clear mucus via ciliary activity to the mouth. Thereduction in mechanical clearance of mucus from the lung leads tochronic bacterial colonization of mucus adherent to airway surfaces. Itis the chronic retention of bacteria, the failure of local antimicrobialsubstances to kill mucus-entrapped bacteria on a chronic basis, and theconsequent chronic inflammatory responses of the body to this type ofsurface infection, that lead to the syndromes of CB and CF.

The current afflicted population in the U.S. is 12,000,000 patients withthe acquired (primarily from cigarette smoke exposure) form of chronicbronchitis and approximately 30,000 patients with the genetic form,cystic fibrosis. Approximately equal numbers of both populations arepresent in Europe. In Asia, there is little CF but the incidence of CBis high and, like the rest of the world, is increasing.

There is currently a large, unmet medical need for products thatspecifically treat CB and CF at the level of the basic defect that causethese diseases. The current therapies for chronic bronchitis and cysticfibrosis focus on treating the symptoms and/or the late effects of thesediseases. Thus, for chronic bronchitis, β-agonists, inhaled steroids,anti-cholinergic agents, and oral theophyllines and phosphodiesteraseinhibitors are all in development. However, none of these drugs treateffectively the fundamental problem of the failure to clear mucus fromthe lung. Similarly, in cystic fibrosis, the same spectrum ofpharmacologic agents is used. These strategies have been complemented bymore recent strategies designed to clear the CF lung of the DNA(“Pulmozyme”; Genentech) that has been deposited in the lung byneutrophils that have futilely attempted to kill the bacteria that growin adherent mucus masses and through the use of inhaled antibiotics(“TOBI”) designed to augment the lungs' own killing mechanisms to ridthe adherent mucus plaques of bacteria. A general principle of the bodyis that if the initiating lesion is not treated, in this case mucusretention/obstruction, bacterial infections became chronic andincreasingly refractory to antimicrobial therapy. Thus, a major unmettherapeutic need for both CB and CF lung diseases is an effective meansof re-hydrating airway mucus (i.e., restoring/expanding the volume ofthe ASL) and promoting its clearance, with bacteria, from the lung.

R. C. Boucher, in U.S. Pat. No. 6,264,975, describes the use ofpyrazinoylguanidine sodium channel blockers for hydrating mucosalsurfaces. These compounds, typified by the well-known diureticsamiloride, benzamil, and phenamil, are effective. However, thesecompounds suffer from the significant disadvantage that they are (1)relatively impotent, which is important because the mass of drug thatcan be inhaled by the lung is limited; (2) rapidly absorbed, whichlimits the half-life of the drug on the mucosal surface; and (3) arefreely dissociable from ENaC. The sum of these disadvantages embodied inthese well known diurectics produces compounds with insufficient potencyand/or effective half-life on mucosal surfaces to have therapeuticbenefit for hydrating mucosal surfaces.

Clearly, what is needed are drugs that are more effective at restoringthe clearance of mucus from the lungs of patients with CB/CF. The valueof these new therapies will be reflected in improvements in the qualityand duration of life for both the CF and the CB populations.

Other mucosal surfaces in and on the body exhibit subtle differences inthe normal physiology of the protective surface liquids on theirsurfaces but the pathophysiology of disease reflects a common theme,i.e., too little protective surface liquid. For example, in xerostomia(dry mouth) the oral cavity is depleted of liquid due to a failure ofthe parotid sublingual and submandibular glands to secrete liquiddespite continued Na⁺ (ENaC) transport mediated liquid absorption fromthe oral cavity. Similarly, keratoconjunctivitis sira (dry eye) iscaused by failure of lacrimal glands to secrete liquid in the face ofcontinued Na⁺ dependent liquid absorption on conjunctional surfaces. Inrhinosinusitis, there is an imbalance, as in CB, between mucin secretionand relative ASL depletion. Finally, in the gastrointestinal tract,failure to secrete Cl⁻ (and liquid) in the proximal small intestine,combined with increased Na⁺ (and liquid) absorption in the terminalileum leads to the distal intestinal obstruction syndrome (DIOS). Inolder patients excessive Na⁺ (and volume) absorption in the descendingcolon produces constipation and diverticulitis.

Fifty million Americans and hundreds of millions of others around theworld suffer from high blood pressure and the subsequent sequale leadingto congestive heart failure and increasing mortality. It is the WesternWorld's leading killer and there is a need there for new medicines totreat these diseases. Thus, in addition, some of the novel sodiumchannel blockers of this invention can be designed to target the kidneyand as such they may be used as diuretics for the treatment ofhypertension, congestive heart failure (CHF) and other cardiovasculardiseases. These new agents may be used alone or in combination withbeta-blockers, ACE inhibitors, HMGCoA reductase inhibitors, calciumchannel blockers and other cardiovascular agents.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide compounds that aremore potent and/or absorbed less rapidly from mucosal surfaces, and/orare less reversible as compared to known compounds.

It is another aspect of the present invention to provide compounds thatare more potent and/or absorbed less rapidly and/or exhibit lessreversibility, as compared to compounds such as amilorde, benzamil, andphenamil. Therefore, the compounds will give a prolonged pharmacodynamichalf-life on mucosal surfaces as compared to known compounds.

It is another object of the present invention to provide compounds whichare (1) absorbed less rapidly from mucosal surfaces, especially airwaysurfaces, as compared to known compounds and; (2) when absorbed frommusosal surfaces after administration to the mucosal surfaces, areconverted in vivo into metabolic derivitives thereof which have reducedefficacy in blocking sodium channels as compared to the administeredparent compound.

It is another object of the present invention to provide compounds thatare more potent and/or absorbed less rapidly and/or exhibit lessreversibility, as compared to compounds such as amiloride, benzamil, andphenamil. Therefore, such compounds will give a prolongedpharmacodynamic half-life on mucosal surfaces as compared to previouscompounds.

It is another object of the present invention to provide compounds thattarget the kidney for use in the treatment of cardiovascular disease.

It is another object of the present invention to provide methods oftreatment that take advantage of the pharmacological properties of thecompounds described above.

In particular, it is an object of the present invention to providemethods of treatment which rely on rehydration of mucosal surfaces.

In particular, it is an object of the present invention to providemethods of treating cardiovascular disease.

The objects of the present invention may be accomplished with a class ofpyrazinoylguanidine compounds represented by formula (I):

where

X is hydrogen, halogen, trifluoromethyl, lower alkyl, unsubstituted orsubstituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio, loweralkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl;

Y is hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio,halogen, lower alkyl, unsubstituted or substituted mononuclear aryl, or—N(R²)₂;

R¹ is hydrogen or lower alkyl;

each R² is, independently, —R⁷, —(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-Z_(g)—R⁷, —(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

where when two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect toeach other the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane;

R³ and R⁴ are each, independently, hydrogen, a group represented byformula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-loweralkyl, (halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, with the proviso that at least one of R³ and R⁴ is a grouprepresented by formula (A):

where

each R^(L) is, independently, —R⁷, —(CH₂)_(n)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸)_(n)—(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷, —(CH₂), —NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂), —CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

where when two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect toeach other the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane;

-   -   each o is, independently, an integer from 0 to 10;    -   each p is an integer from 0 to 10;    -   with the proviso that the sum of o and p in each contiguous        chain is from 1 to 10;    -   each x is, independently, O, NR¹⁰, C(═O), CHOH, C(═N—R¹⁰),        CHNR⁷R¹⁰, or represents a single bond;    -   each R⁵ is, independently, —(CH₂)_(n)—NR¹²R¹²,        —O—(CH₂)_(m)—NR¹²R¹², —O—(CH₂)_(n)—NR¹²R¹²,        —O—(CH₂)_(m)-(Z)_(g)R¹², —(CH₂)_(n)NR¹¹R¹¹, —O—(CH₂)_(m)NR¹¹R¹¹,        —(CH₂)_(n)—N^(⊕)—(R¹¹)₃, —O—(CH₂)_(m)—N^(⊕)—(R¹¹)₃,        —(CH₂)_(n)-(Z)_(g)-(CH₂)_(m)—NR¹⁰R¹⁰,        —O—(CH₂)_(m)-(Z)_(g)-(CH₂)_(m)—NR¹⁰R¹⁰,        —(CH₂CH₂O)_(m)—CH₂CH₂NR¹²R¹², —O—(CH₂CH₂O)_(m)—CH₂CH₂NR¹²R¹²,        —(CH₂)_(n)—(C═O)NR¹²R¹², —O—(CH₂)_(m)—(C═O)NR¹²R¹²,        —O—(CH₂)_(m)—(CHOR⁸)_(m)CH₂NR¹⁰-(Z)_(g)-R¹⁰,        —(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹⁰-(Z)_(g)-R¹⁰,        —(CH₂)_(n)NR¹⁰—O(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹⁰-(Z)_(g)-R¹⁰,        —O(CH₂)_(m)—NR¹⁰—(CH₂)_(m)—(CHOR⁸)_(n)CH₂NR¹⁰-(Z)_(g)-R¹⁰,        -(Het)-(CH₂)_(m)—OR⁸, -(Het)-(CH₂)_(m)—NR⁷R¹⁰,        -(Het)-(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,        -(Het)-(CH₂CH₂O)_(m)—R⁸, -(Het)-(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,        -(Het)-(CH₂)_(m)—C(═O)NR⁷R¹⁰, -(Het)-(CH₂)_(m)-(Z)_(g)-R⁷,        -(Het)-(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,        -(Het)-(CH₂)_(m)—CO₂R⁷, -(Het)-(CH₂)_(m)—NR¹²R¹²,        -(Het)-(CH₂)_(n)—NR¹²R¹², -(Het)-(CH₂)_(m)-(Z)_(g)R¹²,        -(Het)-(CH₂)_(m)NR¹¹R¹¹, -(Het)-(CH₂)_(m)—N^(⊕)—(R¹¹)₃,        -(Het)-(CH₂)_(m)-(Z)_(g)-(CH₂)_(m)—NR¹⁰R¹⁰,        -(Het)-(CH₂CH₂O)_(m)—CH₂CH₂NR¹²R¹²,        -(Het)-(CH₂)_(m)—(C═O)NR¹²R¹²,        -(Het)-(CH₂)_(m)—(CHOR⁸)_(m)CH₂NR¹⁰-(Z)_(g)-R¹⁰,        -(Het)-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)—(CHOR⁸)_(n)CH₂NR¹⁰-(Z)_(g)-R¹⁰,        where when two —CH₂OR⁸ groups are located 1,2- or 1,3- with        respect to each other the R⁸ groups may be joined to form a        cyclic mono- or di-substituted 1,3-dioxane or 1,3-dioxolane,

—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸ with the proviso that two —CH₂OR⁸groups are located 1,2- or 1,3- with respect to each other and the R⁸groups are joined to form a cyclic mono or disubstituted 1,3-dioxane or1,3-dioxolane,

—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸ with the proviso that two —CH₂OR⁸groups are located 1,2- or 1,3- with respect to each other and the R⁸groups are joined to form a cyclic mono or disubstituted 1,3-dioxane or1,3-dioxolane,

—(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with the proviso that two—CH₂OR⁸ groups are located 1,2- or 1,3- with respect to each other andthe R⁸ groups are joined to form a cyclic mono or disubstituted1,3-dioxane or 1,3-dioxolane, or

—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with the proviso thattwo —CH₂OR⁸ groups are located 1,2- or 1,3- with respect to each otherand the R⁸ groups are joined to form a cyclic mono or disubstituted1,3-dioxane or 1,3-dioxolane;

each R⁶ is, independently, —R⁵, —R⁷, —OR⁸, —N(R⁷)₂, —(CH₂)_(m)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

where when two R⁶ are —OR¹¹ and are located adjacent to each other on aphenyl ring, the alkyl moieties of the two R⁶ may be bonded together toform a methylenedioxy group and

where when two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect toeach other the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane;

-   -   each R⁷ is, independently, hydrogen or lower alkyl;    -   each R⁸ is, independently, hydrogen, lower alkyl, —C(═O)—R¹¹,        glucuronide, 2-tetrahydropyranyl, or

-   -   each R⁹ is, independently, —CO₂R⁷, —CON(R⁷)₂, —SO₂CH₃, or        —C(═O)R⁷;    -   each R¹⁰ is, independently, —H, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹,        —C(═O)R⁷, or —CH₂—(CHOH)_(n)—CH₂OH;    -   each Z is, independently, CHOH, C(═O), CHNR⁷R¹⁰, C═NR¹⁰, or        NR¹⁰;    -   each R¹¹ is, independently, lower alkyl;    -   each R¹² is independently, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹,        —C(═O)R⁷, or —CH₂—(CHOH)_(n)—CH₂OH;    -   each Het is independently, —NR⁷, —NR¹⁰, —S—, —SO—, or —SO₂—;    -   each g is, independently, an integer from 1 to 6;    -   each m is, independently, an integer from 1 to 7;    -   each n is, independently, an integer from 0 to 7;    -   each Q is, independently, C—R⁵, C—R⁶, or a nitrogen atom,        wherein at most three Q in a ring are nitrogen atoms;

or a pharmaceutically acceptable salt thereof, and

inclusive of all enantiomers, diastereomers, and racemic mixturesthereof.

The present also provides pharmaceutical compositions which contain acompound described above.

The present invention also provides a method of promoting hydration ofmucosal surfaces, comprising:

administering an effective amount of a compound represented by formula(I) to a mucosal surface of a subject.

The present invention also provides a method of restoring mucosaldefense, comprising:

topically administering an effective amount of compound represented byformula (I) to a mucosal surface of a subject in need thereof.

The present invention also provides a method of blocking ENaC,comprising:

contacting sodium channels with an effective amount of a compoundrepresented by formula (I).

The present invention also provides a method of promoting mucusclearance in mucosal surfaces, comprising:

administering an effective amount of a compound represented by formula(I) to a mucosal surface of a subject.

The present invention also provides a method of treating chronicbronchitis, comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating cysticfibrosis, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating rhinosinusitis,comprising:

administering an effective amount of a compound represented by a formula(I) to a subject in need thereof.

The present invention also provides a method of treating nasaldehydration, comprising:

administering an effective amount of a compound represented by formula(I) to the nasal passages of a subject in need thereof.

In a more specific embodiment, the nasal dehydration is brought on byadministering dry oxygen to the subject.

The present invention also provides a method of treating sinusitis,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating pneumonia,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of preventingventilator-induced pneumonia, comprising:

administering an effective compound represented by formula (I) to asubject by means of a ventilator.

The present invention also provides a method of treating asthma,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating primary ciliarydyskinesia, comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating otitis media,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of inducing sputum fordiagnostic-purposes, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating chronicobstructive pulmonary disease, comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating emphysema,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating dry eye,comprising:

administering an effective amount of a compound represented by formula(I) to the eye of the subject in need thereof.

The present invention also provides a method of promoting ocularhydration, comprising:

administering an effective amount of a compound represented by formula(I) to the eye of the subject.

The present invention also provides a method of promoting cornealhydration, comprising:

administering an effective amount of a compound represented by formula(I) to the eye of the subject.

The present invention also provides a method of treating Sjögren'sdisease, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating vaginaldryness, comprising:

administering an effective amount of a compound represented by formula(I) to the vaginal tract of a subject in need thereof.

The present invention also provides a method of treating dry skin,comprising:

administering an effective amount of a compound represented by formula(I) to the skin of a subject in need thereof.

The present invention also provides a method of treating dry mouth(xerostomia), comprising:

administering an effective amount of compound represented by formula (I)to the mouth of the subject in need thereof.

The present invention also provides a method of treating distalintestinal obstruction syndrome, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating esophagitis,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating constipation,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof. In one embodiment of this method, thecompound is administered either orally or via a suppository or enema.

The present invention also provides a method of treating chronicdiverticulitis comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating hypertension,comprising administering the compound represented by formula (D) to asubject in need thereof.

The present invention also provides a method of reducing blood pressure,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of treating edema,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of promoting diuresis,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of promoting natriuresis,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of promoting saluresis,comprising administering the compound represented by formula (I) to asubject in need thereof.

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that the compounds offormula (I) are more potent and/or, absorbed less rapidly from mucosalsurfaces, especially airway surfaces, and/or less reversible frominteractions with ENaC as compared to compounds such as amiloride,benzamil, and phenamil. Therefore, the compounds of formula (I) have alonger half-life on mucosal surfaces as compared to these compounds.

The present invention is also based on the discovery that certaincompounds embraced by formula (I) are converted in vivo into metabolicderivitives thereof that have reduced efficacy in blocking sodiumchannels as compared to the parent administered compound, after they areabsorbed from mucosal surfaces after administration. This importantproperty means that the compounds will have a lower tendency to causeundesired side-effects by blocking sodium channels located at untargetedlocations in the body of the recipient, e.g., in the kidneys.

-   The present invention is also based on the discovery that certain    compounds embraced by formula (I) target the kidney and thus may be    used as cardiovascular agents.

In the compounds represented by formula (I), X may be hydrogen, halogen,trifluoromethyl, lower alkyl, lower cycloalkyl, unsubstituted orsubstituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio, loweralkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl. Halogen is preferred.

Examples of halogen include fluorine, chlorine, bromine, and iodine.Chlorine and bromine are the preferred halogens. Chlorine isparticularly preferred. This description is applicable to the term“halogen” as used throughout the present disclosure.

As used herein, the term “lower alkyl” means an alkyl group having lessthan 8 carbon atoms. This range includes all specific values of carbonatoms and subranges there between, such as 1 ,2, 3, 4, 5, 6, and 7carbon atoms. The term “alkyl” embraces all types of such groups, e.g.,linear, branched, and cyclic alkyl groups. This description isapplicable to the term “lower alkyl” as used throughout the presentdisclosure. Examples of suitable lower alkyl groups include methyl,ethyl, propyl, cyclopropyl, butyl, isobutyl, etc.

Substituents for the phenyl group include halogens. Particularlypreferred halogen substituents are chlorine and bromine.

Y may be hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio,halogen, lower alkyl, lower cycloalkyl, mononuclear aryl, or —N(R²)₂.The alkyl moiety of the lower alkoxy groups is the same as describedabove. Examples of mononuclear aryl include phenyl groups. The phenylgroup may be unsubstituted or substituted as described above. Thepreferred identity of Y is —N(R²)₂. Particularly preferred are suchcompounds where each R² is hydrogen.

R¹ may be hydrogen or lower alkyl. Hydrogen is preferred for R¹.

Each R² may be, independently, —R⁷, —(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-Z_(g)-R⁷, —(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷ or

In the definition of R² described above, when two —CH₂OR⁸ groups arelocated 1,2- or 1,3- with respect to each other the R⁸ groups may bejoined to form a cyclic mono- or di-substituted 1,3-dioxane or1,3-dioxolane.

Hydrogen and lower alkyl, particularly C₁-C₃ alkyl are preferred for R².Hydrogen is particularly preferred.

R³ and R⁴ may be, independently, hydrogen, a group represented byformula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-loweralkyl, (halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, provided that at least one of R³ and R⁴ is a group represented byformula (A).

Preferred compounds are those where one of R³ and R⁴ is hydrogen and theother is represented by formula (A).

In formula (A), the moiety —(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)— definesan alkylene group bonded to the aromatic ring. The variables o and p mayeach be an integer from 0 to 10, subject to the proviso that the sum ofo and p in the chain is from 1 to 10. Thus, o and p may each be 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the sum of o and p is from 2 to6. In a particularly preferred embodiment, the sum of o and p is 4.

The linking group in the alkylene chain, x, may be, independently, O,NR¹⁰, C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰, or represents a single bond;

Therefore, when x represents a single bond, the alkylene chain bonded tothe ring is represented by the formula —(C(R^(L))₂)_(o+p)—, in which thesum o+p is from 1 to 10.

Each R^(L) may be, independently, —R⁷, —(CH₂)_(n)—OR⁸—O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

In the definition of R^(L) above, when two —CH₂OR⁸ groups are located1,2- or 1,3- with respect to each other the R⁸ groups may be joined toform a cyclic mono- or di-substituted 1,3-dioxane or 1,3-dioxolane.

The preferred R^(L) groups include —H, —OH, —N(R⁷)₂, especially whereeach R⁷ is hydrogen.

In the alkylene chain in formula (A), it is preferred that when oneR^(L) group bonded to a carbon atoms is other than hydrogen, then theother R^(L) bonded to that carbon atom is hydrogen, i.e., the formula—CHR^(L)—. It is also preferred that at most two R^(L) groups in analkylene chain are other than hydrogen, where in the other R^(L) groupsin the chain are hydrogens. Even more preferably, only one R^(L) groupin an alkylene chain is other than hydrogen, where in the other R^(L)groups in the chain are hydrogens. In these embodiments, it ispreferable that x represents a single bond.

In another particular embodiment of the invention, all of the R^(L)groups in the alkylene chain are hydrogen. In these embodiments, thealkylene chain is represented by the formula—(CH₂)_(o)-x-(CH₂)_(p)—.

Each R⁵ is independently, —(CH₂)_(n)—NR¹²R¹², —O—(CH₂)_(m)—NR¹²R¹²,—O—(CH₂)_(n)—NR¹²R¹², —O—(CH₂)_(m)-(Z)_(g)R¹², —(CH₂)_(n)NR¹¹R¹¹,—O—(CH₂)_(m)NR¹¹R¹¹, —(CH₂)_(n)—N^(⊕)—(R¹¹)₃, —O—(CH₂)_(m)—N^(⊕)—(R¹¹)₃,—(CH₂)_(n)-(Z)_(g)-(CH₂)_(m)—NR¹⁰R¹⁰,—O—(CH₂)_(m)-(Z)_(g)-(CH₂)_(m)—NR¹⁰R¹⁰, —(CH₂CH₂O)_(m)—CH₂CH₂NR¹²R¹²,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR¹²R¹², —(CH₂)_(n)—(C═O)NR¹²R¹²,—O—(CH₂)_(m)—(C═O)NR¹²R¹², —O—(CH₂)_(m)—(CHOR⁸)_(m)CH₂NR¹⁰-(Z)_(g)-R¹⁰,—(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹⁰-(Z)_(g)-R¹⁰,—(CH₂)_(n)NR¹⁰—O(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹⁰-(Z)_(g)-R¹⁰,—O(CH₂)_(m)—NR¹⁰—(CH₂)_(m)—(CHOR⁸)_(n)CH₂NR¹⁰-(Z)_(g)-R¹⁰,-(Het)-(CH₂)_(m)—OR⁸, -(Het)-(CH₂)_(m)—NR⁷R¹⁰,-(Het)-(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, -(Het)-(CH₂CH₂O)_(m)—R⁸,-(Het)-(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, -(Het)-(CH₂)_(m)—C(═O)NR⁷R¹⁰,-(Het)-(CH₂)_(m)-(Z)_(g)-R⁷,-(Het)-(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,-(Het)-(CH₂)_(m)—CO₂R⁷, -(Het)-(CH₂)_(m)—NR¹²R¹²,-(Het)-(CH₂)_(n)—N¹²R¹², -(Het)-(CH₂)_(m)-(Z)_(g)R¹²,-(Het)-(CH₂)_(m)NR¹¹R¹¹, -(Het)-(CH₂)_(m)—N^(⊕)—(R¹¹)₃,-(Het)-(CH₂)_(m)-(Z)_(g)-(CH₂)_(m)—NR¹⁰R¹⁰,-(Het)-(CH₂CH₂O)_(m)—CH₂CH₂NR¹²R¹², -(Het)-(CH₂)_(m)—(C═O)NR¹²R¹²,-(Het)-(CH₂)_(m)—(CHOR⁸)_(m)CH₂NR¹⁰-(Z)_(g)-R¹⁰,-(Het)-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)—(CHOR⁸)_(n)CH₂NR¹⁰-(Z)_(g)-R¹⁰, wherewhen two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect to eachother the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane,

—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with the proviso that two —CH₂OR⁸groups are located 1,2- or 1,3- with respect to each other and the R⁸groups are joined to form a cyclic mono or disubstituted 1,3-dioxane or1,3-dioxolane,

—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with the proviso that two —CH₂OR⁸groups are located 1,2- or 1,3- with respect to each other and the R⁸groups are joined to form a cyclic mono or disubstituted 1,3-dioxane or1,3-dioxolane,

—(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with the proviso that two—CH₂OR⁸ groups are located 1,2- or 1,3- with respect to each other andthe R⁸ groups are joined to form a cyclic mono or disubstituted1,3-dioxane or 1,3-dioxolane, or

—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with the proviso thattwo —CH₂OR⁸ groups are located 1,2- or 1,3- with respect to each otherand the R⁸ groups are joined to form a cyclic mono or disubstituted1,3-dioxane or 1,3-dioxolane.

Preferred examples of R⁵ include:

—N(SO₂CH₃)₂,

—CH₂—CHNHBocCO₂CH₃ (α),

—O—CH₂—CHNH₂CO₂H (α),

—O—CH₂—CHNH₂CO₂CH₃ (α),

—O—(CH₂)₂—N⁺(CH₃)₃,

—C(═O)NH—(CH₂)₂—NH₂,

—C(═O)NH—(CH₂)₂—NH—C(═NH)—NH₂, and

There are four R⁶ groups present on the ring in formula (A). Each R⁶ maybe each, independently, R⁵ as described above, —R⁷, —OR⁸, —N(R⁷)₂,—(CH₂)_(m)—OR⁸, —O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰,—O—(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷, —(CH₂), —NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose, or

When two R⁶ are —OR¹¹ and are located adjacent to each other on a phenylring, the alkyl moieties of the two R⁶ groups may be bonded together toform a methylenedioxy group, i.e., a group of the formula —O—CH₂—O—.Also, when two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect toeach other the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane.

As discussed above, R⁶ may be hydrogen. Therefore, 1, 2, 3, or 4 R⁶groups may be other than hydrogen. Preferably at most 3 of the R⁶ groupsare other than hydrogen.

Each g is, independently, an integer from 1 to 6. Therefore, each g maybe 1, 2, 3, 4, 5, or 6.

Each m is an integer from 1 to 7. Therefore, each m may be 1, 2, 3, 4,5, 6, or 7.

Each n is an integer from 0 to 7. Therefore, each n maybe 0, 1, 2, 3, 4,5, 6, or 7.

Each Q in formula (A) is C—R⁵, C—R⁶, or a nitrogen atom, where at mostthree Q in a ring are nitrogen atoms. Thus, there may be 1, 2, or 3nitrogen atoms in a ring. Preferably, at most two Q are nitrogen atoms.More preferably, at most one Q is a nitrogen atom. In one particularembodiment, the nitrogen atom is at the 3-position of the ring. Inanother embodiment of the invention, each Q is either C—R⁵ or C—R⁶,i.e., there are no nitrogen atoms in the ring.

More specific examples of suitable groups represented by formula (A) areshown in formulas (B)-(F) below:

where o, x, p, R⁵, and R⁶, are as defined above;

where n is an integer from 1 to 10 and R⁵ is as defined above;

where n is an integer from 1 from 10 and R⁵ is as defined above;

where o, x, p, and R⁵ are as defined above;

In a preferred embodiment of the invention, Y is —NH₂.

In another preferred embodiment, R² is hydrogen.

In another preferred embodiment, R¹ is hydrogen.

In another preferred embodiment, X is chlorine.

In another preferred embodiment, R³ is hydrogen.

In another preferred embodiment, R^(L) is hydrogen.

In another preferred embodiment, o is 4.

In another preferred embodiment, p is 0.

In another preferred embodiment, the sum of o and p is 4.

In another preferred embodiment, x represents a single bond.

In another preferred embodiment, R⁶ is hydrogen.

In another preferred embodiment, at most one Q is a nitrogen atom.

In another preferred embodiment, no Q is a nitrogen atom.

In a preferred embodiment of the present invention:

X is halogen;

Y is —N(R⁷)₂;

R¹ is hydrogen or C₁-C₃ alkyl;

R² is —R⁷, —OR⁷, CH₂OR⁷, or —CO₂R⁷;

R³ is a group represented by formula (A); and

R⁴ is hydrogen, a group represented by formula (A), or lower alkyl;

In another preferred embodiment of the present invention:

X is chloro or bromo;

Y is —N(R⁷)₂;

R² is hydrogen or C₁-C₃ alkyl;

at most three R⁶ are other than hydrogen as described above;

at most three R^(L) are other than hydrogen as described above; and

at most 2 Q are nitrogen atoms.

In another preferred embodiment of the present invention:

Y is —NH₂;

In another preferred embodiment of the present invention:

R⁴ is hydrogen;

at most one R^(L) is other than hydrogen as described above;

at most two R⁶ are other than hydrogen as described above; and

at most 1 Q is a nitrogen atom.

Preferred examples of R⁵ in the embodiments described above include:

—N(SO₂CH₃)₂,

—CH₂—CHNHBocCO₂CH₃ (α),

—O—CH₂—CHNH₂CO₂H (α),

—O—CH₂—CHNH₂CO₂CH₃ (α),

—O—(CH₂)₂—N⁺(CH₃)₃,

—C(═O)NH—(CH₂)₂—NH₂, and

—C(═O)NH—(CH₂)₂—NH—C(═NH)—NH₂.

Examples of compounds of the present invention include the following:

The compounds of formula (I) may be prepared and used as the free base.Alternatively, the compounds may be prepared and used as apharmaceutically acceptable salt. Pharmaceutically acceptable salts aresalts that retain or enhance the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; (b) salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolicacid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid,phthalic acid, mandelic acid, lactic acid and the like; and (c) saltsformed from elemental anions for example, chlorine, bromine, and iodine.

It is to be noted that all enantiomers, diastereomers, and racemicmixtures of compounds within the scope of formula (I) are embraced bythe present invention. All mixtures of such enantiomers anddiastereomers are within the scope of the present invention.

Without being limited to any particular theory, it is believed that thecompounds of formula (I) function in vivo as sodium channel blockers. Byblocking epithelial sodium channels present in mucosal surfaces thecompounds of formula (L) reduce the absorption of water by the mucosalsurfaces. This effect increases the volume of protective liquids onmucosal surfaces, rebalances the system, and thus treats disease.

The present invention also provides methods of treatment that takeadvantage of the properties of the compounds of formula (I) discussedabove. Thus, subjects that may be treated by the methods of the presentinvention include, but are not limited to, patients afflicted withcystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, chronicobstructive airway disease, artificially ventilated patients, patientswith acute pneumonia, etc. The present invention may be used to obtain asputum sample from a patient by administering the active compounds to atleast one lung of a patient, and then inducing or collecting a sputumsample from that patient. Typically, the invention will be administeredto respiratory mucosal surfaces via aerosol (liquid or dry powders) orlavage.

Subjects that may be treated by the method of the present invention alsoinclude patients being administered supplemental oxygen nasally (aregimen that tends to dry the airway surfaces); patients afflicted withan allergic disease or response (e.g., an allergic response to pollen,dust, animal hair or particles, insects or insect particles, etc.) thataffects nasal airway surfaces; patients afflicted with a bacterialinfection e.g., staphylococcus infections such as Staphylococcus aureusinfections, Hemophilus influenza infections, Streptococcus pneumoniaeinfections, Pseudomonas aeuriginosa infections, etc.) of the nasalairway surfaces; patients afflicted with an inflammatory disease thataffects nasal airway surfaces; or patients afflicted with sinusitis(wherein the active agent or agents are administered to promote drainageof congested mucous secretions in the sinuses by administering an amounteffective to promote drainage of congested fluid in the sinuses), orcombined, Rhinosinusitis. The invention may be administered torhino-sinal surfaces by topical delivery, including aerosols and drops.

The present invention may be used to hydrate mucosal surfaces other thanairway surfaces. Such other mucosal surfaces include gastrointestinalsurfaces, oral surfaces, genitourethral surfaces, ocular surfaces orsurfaces of the eye, the inner ear and the middle ear. For example, theactive compounds of the present invention may be administered by anysuitable means, including locally/topically, orally, or rectally, in aneffective amount.

The compounds of the present invention are also useful for treating avariety of functions relating to the cardiovascular system. Thus, thecompounds of the present invention are useful for use asantihypertensive agents. The compounds may also be used to reduce bloodpressure and to treat edema. In addition, the compounds of the presentinvention are also useful for promoting diuresis, natriuresis, andsaluresis. The compounds may be used alone or in combination with betablockers, ACE inhibitors, HMGCoA reductase inhibitors, calcium channelblockers and other cardiovascular agents to treat hypertension,congestive heart failure and reduce cardiovascular mortality.

The present invention is concerned primarily with the treatment of humansubjects, but may also be employed for the treatment of other mammaliansubjects, such as dogs and cats, for veterinary purposes.

As discussed above, the compounds used to prepare the compositions ofthe present invention may be in the form of a pharmaceuticallyacceptable free base. Because the free base of the compound is generallyless soluble in aqueous solutions than the salt, free base compositionsare employed to provide more sustained release of active agent to thelungs. An active agent present in the lungs in particulate form whichhas not dissolved into solution is not available to induce aphysiological response, but serves as a depot of bioavailable drug whichgradually dissolves into solution.

Another aspect of the present invention is a pharmaceutical composition,comprising a compound of formula (I) in a pharmaceutically acceptablecarrier (e.g., an aqueous carrier solution). In general, the compound offormula (I) is included in the composition in an amount effective toinhibit the reabsorption of water by mucosal surfaces.

The compounds of the present invention may also be used in conjunctionwith a P2Y2 receptor agonist or a pharmaceutically acceptable saltthereof (also sometimes referred to as an “active agent” herein). Thecomposition may further comprise a P2Y2 receptor agonist or apharmaceutically acceptable salt thereof (also sometimes referred to asan “active agent” herein). The P2Y2 receptor agonist is typicallyincluded in an amount effective to stimulate chloride and watersecretion by airway surfaces, particularly nasal airway surfaces.Suitable P2Y2 receptor agonists are described in columns 9-10 of U.S.Pat. Nos. 6,264,975, 5,656,256, and 5,292,498, each of which isincorporated herein by reference.

Bronchodiloators can also be used in combination with compounds of thepresent invention. These bronchodilators include, but are not limitedto, β-adrenergic agonists including but not limited to epinephrine,isoproterenol, fenoterol, albutereol, terbutalin, pirbuterol,bitolterol, metaproterenol, iosetharine, salmeterol xinafoate, as wellas anticholinergic agents including but not limited to ipratropiumbromide, as well as compounds such as theophylline and aminophylline.These compounds may be administered in accordance with known techniques,either prior to or concurrently with the active compounds describedherein.

Another aspect of the present invention is a pharmaceutical formulation,comprising an active compound as described above in a pharmaceuticallyacceptable carrier (e.g., an aqueous carrier solution). In general, theactive compound is included in the composition in an amount effective totreat mucosal surfaces, such as inhibiting the reabsorption of water bymucosal surfaces, including airway and other surfaces.

The active compounds disclosed herein may be administered to mucosalsurfaces by any suitable means, including topically, orally, rectally,vaginally, ocularly and dermally, etc. For example, for the treatment ofconstipation, the active compounds may be administered orally orrectally to the gastrointestinal mucosal surface. The active compoundmay be combined with a pharmaceutically acceptable carrier in anysuitable form, such as sterile physiological or dilute saline or topicalsolution, as a droplet, tablet or the like for oral administration, as asuppository for rectal or genito-urethral administration, etc.Excipients may be included in the formulation to enhance the solubilityof the active compounds, as desired.

The active compounds disclosed herein may be administered to the airwaysurfaces of a patient by any suitable means, including as a spray, mist,or droplets of the active compounds in a pharmaceutically acceptablecarrier such as physiological or dilute saline solutions or distilledwater. For example, the active compounds may be prepared as formulationsand administered as described in U.S. Pat. No. 5,789,391 to Jacobus, thedisclosure of which is incorporated by reference herein in its entirety.

Solid or liquid particulate active agents prepared for practicing thepresent invention could, as noted above, include particles of respirableor non-respirable size; that is, for respirable particles, particles ofa size sufficiently small to pass through the mouth and larynx uponinhalation and into the bronchi and alveoli of the lungs, and fornon-respirable particles, particles sufficiently large to be retained inthe nasal airway passages rather than pass through the larynx and intothe bronchi and alveoli of the lungs. In general, particles ranging fromabout 1 to 5 microns in size (more particularly, less than about 4.7microns in size) are respirable. Particles of non-respirable size aregreater than about 5 microns in size, up to the size of visibledroplets. Thus, for nasal administration, a particle size in the rangeof 10-500 μm may be used to ensure retention in the nasal cavity.

In the manufacture of a formulation according to the invention, activeagents or the physiologically acceptable salts or free bases thereof aretypically admixed with, inter alia, an acceptable carrier. Of course,the carrier must be compatible with any other ingredients in theformulation and must not be deleterious to the patient. The carrier mustbe solid or liquid, or both, and is preferably formulated with thecompound as a unit-dose formulation, for example, a capsule, that maycontain 0.5% to 99% by weight of the active compound. One or more activecompounds may be incorporated in the formulations of the invention,which formulations may be prepared by any of the well-known techniquesof pharmacy consisting essentially of admixing the components.

Compositions containing respirable or non-respirable dry particles ofmicronized active agent may be prepared by grinding the dry active agentwith a mortar and pestle, and then passing the micronized compositionthrough a 400 mesh screen to break up or separate out largeagglomerates.

The particulate active agent composition may optionally contain adispersant which serves to facilitate the formulation of an aerosol. Asuitable dispersant is lactose, which may be blended with the activeagent in any suitable ratio (e.g., a 1 to 1 ratio by weight).

Active compounds disclosed herein may be administered to airway surfacesincluding the nasal passages, sinuses and lungs of a subject by asuitable means know in the art, such as by nose drops, mists, etc. Inone embodiment of the invention, the active compounds of the presentinvention and administered by transbronchoscopic lavage. In a preferredembodiment of the invention, the active compounds of the presentinvention are deposited on lung airway surfaces by administering anaerosol suspension of respirable particles comprised of the activecompound, which the subject inhales. The respirable particles may beliquid or solid. Numerous inhalers for administering aerosol particlesto the lungs of a subject are known.

Inhalers such as those developed by Inhale Therapeutic Systems, PaloAlto, Calif., USA, may be employed, including but not limited to thosedisclosed in U.S. Pat. Nos. 5,740,794; 5,654,007; 5,458,135; 5,775,320;and 5,785,049, all of which are incorporated herein by reference. TheApplicant specifically intends that the disclosures of all patentreferences cited herein be incorporated by reference herein in theirentirety. Inhalers such as those developed by Dura Pharmaceuticals,Inc., San Diego, Calif., USA, may also be employed, including but notlimited to those disclosed in U.S. Pat. Nos. 5,622,166; 5,577,497;5,645,051; and 5,492,112, all of which are incorporated herein byreference. Additionally, inhalers such as those developed by AradigmCorp., Hayward, Calif., USA, may be employed, including but not limitedto those disclosed in U.S. Pat. Nos. 5,826,570; 5,813,397; 5,819,726;and 5,655,516, all of which are incorporated herein by reference. Theseapparatuses are particularly suitable as dry particle inhalers.

Aerosols of liquid particles comprising the active compound may beproduced by any suitable means, such as with a pressure-driven aerosolnebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No.4,501,729, the contents of which is incorporated herein by reference.Nebulizers are commercially available devices which transform solutionsor suspensions of the active ingredient into a therapeutic aerosol misteither by means of acceleration of compressed gas, typically air oroxygen, through a narrow venturi orifice or by means of ultrasonicagitation. Suitable formulations for use in nebulizers consist of theactive ingredient in a liquid carrier, the active ingredient comprisingup to 40% w/w of the formulation, but preferably less than 20% w/w. Thecarrier is typically water (and most preferably sterile, pyrogen-freewater) or dilute aqueous alcoholic solution. Perfluorocarbon carriersmay also be used. Optional additives include preservatives if theformulation is not made sterile, for example, methyl hydroxybenzoate,antioxidants, flavoring agents, volatile oils, buffering agents, andsurfactants.

Aerosols of solid particles comprising the active compound may likewisebe produced with any solid particulate medicament aerosol generator.Aerosol generators for administering solid particulate medicaments to asubject produce particles which are respirable, as explained above, andgenerate a volume of aerosol containing predetermined metered dose ofmedicament at a rate suitable for human administration. One illustrativetype of solid particulate aerosol generator is an insufflator. Suitableformulations for administration by insufflation include finelycomminuted powders which may be delivered by means of an insufflator ortaken into the nasal cavity in the manner of a snuff. In theinsufflator, the powder (e.g., a metered dose thereof effective to carryout the treatments described herein) is contained in capsules orcartridges, typically made of gelatin or plastic, which are eitherpierced or opened in situ and the powder delivered by air drawn throughthe device upon inhalation or by means of a manually-operated pump. Thepowder employed in the insufflator consists either solely of the activeingredient or of powder blend comprising the active ingredient, asuitable powder diluent, such as lactose, and an optional surfactant.The active ingredient typically comprises of 0.1 to 100% w/w of theformulation. A second type of illustrative aerosol generator comprises ametered dose inhaler. Metered dose inhalers are pressurized aerosoldispensers, typically containing a suspension or solution formulation ofactive ingredient in a liquified propellant. During use, these devicesdischarge the formulation through a valve adapted to deliver a meteredvolume, typically from 10 to 150 μl, to produce a fine particle spraycontaining the active ingredient. Suitable propellants include certainchlorofluorocarbon compounds, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.The formulation may additionally contain one of more co-solvents, forexample, ethanol, surfactants, such as oleic acid or sorbitan trioleate,antioxidants and suitable flavoring agents.

The aerosol, whether formed from solid or liquid particles, may beproduced by the aerosol generator at a rate of from about 10 to 150liters per minute, more preferable from 30 to 150 liters per minute, andmost preferably about 60 liters per minute. Aerosols containing greateramounts of medicament may be administered more rapidly.

The dosage of the active compounds disclosed herein will vary dependingon the condition being treated and the state of the subject, butgenerally may be from about 0.01, 0.03, 0.05, 0.1 to 1, 5, 10 or 20 mgof the pharmaceutic agent, deposited on the airway surfaces. The dailydose may be divided among one or multiple unit dose administrations. Thegoal is to achieve a concentration of the pharmaceutic agents on lungairway surfaces of between 10⁻⁹-10⁴ M.

In another embodiment, they are administered by administering an aerosolsuspension of respirable or non-respirable particles (preferablynon-respirable particles) comprised of active compound, which thesubject inhales through the nose. The respirable or non-respirableparticles may be liquid or solid. The quantity of active agent includedmay be an amount of sufficient to achieve dissolved concentrations ofactive agent on the airway surfaces of the subject of from about 10⁻⁹,10⁻⁸, or 10⁻⁷ to about 10⁻³, 10⁻², 10⁻¹ moles/liter, and more preferablyfrom about 10⁻⁹ to about 10⁻⁴ moles/liter.

The dosage of active compound will vary depending on the condition beingtreated and the state of the subject, but generally may be an amountsufficient to achieve dissolved concentrations of active compound on thenasal airway surfaces of the subject from about 10⁻⁹, 10⁻⁸, or 10⁻⁷ toabout 10⁻³, 10⁻², or 10⁻¹ Moles/liter, and more preferably from about10⁻⁷ to about 10⁻⁴ Moles/liter. Depending upon the solubility of theparticular formulation of active compound administered, the daily dosemay be divided among one or several unit dose administrations. The dailydose by weight may range from about 0.01, 0.03, 0.1, 0.5 or 1.0 to 10 or20 milligrams of active agent particles for a human subject, dependingupon the age and condition of the subject. A currently preferred unitdose is about 0.5 milligrams of active agent given at a regimen of 2-10administrations per day. The dosage may be provided as a prepackagedunit by any suitable means (e.g., encapsulating a gelatin capsule).

In one embodiment of the invention, the particulate active agentcomposition may contain both a free base of active agent and apharmaceutically acceptable salt to provide both early release andsustained release of active agent for dissolution into the mucussecretions of the nose. Such a composition serves to provide both earlyrelief to the patient, and sustained relief over time. Sustained relief,by decreasing the number of daily administrations required, is expectedto increase patient compliance with the course of active agenttreatments.

Pharmaceutical formulations suitable for airway administration includeformulations of solutions, emulsions, suspensions and extracts. Seegenerally, J. Naim, Solutions, Emulsions, Suspensions and Extracts, inRemington: The Science and Practice of Pharmacy, chap. 86 (19^(th) ed1995), incorporated herein by reference. Pharmaceutical formulationssuitable for nasal administration may be prepared as described in U.S.Pat. No. 4,389,393 to Schor; U.S. Pat. No. 5,707,644 to Illum; U.S. Pat.No. 4,294,829 to Suzuki; and U.S. Pat. No. 4,835,142 to Suzuki, thedisclosures of which are incorporated by reference herein.

Mists or aerosols of liquid particles comprising the active compound maybe produced by any suitable means, such as by a simple nasal spray withthe active agent in an aqueous pharmaceutically acceptable carrier, suchas a sterile saline solution or sterile water. Administration may bewith a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. Seee.g. U.S. Pat. Nos. 4,501,729 and 5,656,256, each of which isincorporated herein by reference. Suitable formulations for use in anasal droplet or spray bottle or in nebulizers consist of the activeingredient in a liquid carrier, the active ingredient comprising up to40% w/w of the formulation, but preferably less than 20% w/w. Typicallythe carrier is water (and most preferably sterile, pyrogen-free water)or dilute aqueous alcoholic solution, preferably made in a 0.12% to 0.8%solution of sodium chloride. Optional additives include preservatives ifthe formulation is not made sterile, for example, methylhydroxybenzoate, antioxidants, flavoring agents, volatile oils,buffering agents, osmotically active agents (e.g. mannitol, xylitol,erythritol) and surfactants.

Compositions containing respirable or non-respirable dry particles ofmicronized active agent may be prepared by grinding the dry active agentwith a mortar and pestle, and then passing the micronized compositionthrough a 400 mesh screen to break up or separate out largeagglomerates.

The particulate composition may optionally contain a dispersant whichserves to facilitate the formation of an aerosol. A suitable dispersantis lactose, which may be blended with the active agent in any suitableratio (e.g., a 1 to 1 ratio by weight).

The compounds of formula (I) may be synthesized according to proceduresknown in the art. A representative synthetic procedure is shown in thescheme below.

These procedures are described in, for example, E. J. Cragoe, “TheSynthesis of Amiloride and Its Analogs” (Chapter 3) in Amiloride and ItsAnalogs, pp. 25-36, incorporated herein by reference. Other methods ofpreparing the compounds are described in, for example, U.S. Pat. No.3,313,813, incorporated herein by reference. See in particular MethodsA, B, C, and D described in U.S. Pat. No. 3,313,813.

Several assays may be used to characterize the compounds of the presentinvention. Representative assays are discussed below.

In Vitro Measure of Sodium Channel Blocking Activity and Reversibility

One assay used to assess mechanism of action and/or potency of thecompounds of the present invention involves the determination of lumenaldrug inhibition of airway epithelial sodium currents measured undershort circuit current (I_(SC)) using airway epithelial monolayersmounted in Ussing chambers. Cells obtained from freshly excised human,dog, sheep or rodent airways are seeded onto porous 0.4 micron Snapwell™Inserts (CoStar), cultured at air-liquid interface (ALI) conditions inhormonally defined media, and assayed for sodium transport activity(I_(SC)) while bathed in Krebs Bicarbonate Ringer (KBR) in Usingchambers. All test drug additions are to the lumenal bath with half-logdose addition protocols (from 1×10⁻¹¹ M to 3×10⁻⁵M), and the cumulativechange in I_(SC) (inhibition) recorded. All drugs are prepared indimethyl sulfoxide as stock solutions at a concentration of 1×10⁻² M andstored at −20° C. Eight preparations are typically run in parallel; twopreparations per run incorporate amiloride and/or benzamil as positivecontrols. After the maximal concentration (5×10⁻⁵ M) is administered,the lumenal bath is exchanged three times with fresh drug-free KBRsolution, and the resultant I_(SC) measured after each wash forapproximately 5 minutes in duration. Reversibility is defined as thepercent return to the baseline value for sodium current after the thirdwash. All data from the voltage clamps are collected via a computerinterface and analyzed off-line.

Dose-effect relationships for all compounds are considered and analyzedby the Prism 3.0 program. IC₅₀ values, maximal effective concentrations,and reversibility are calculated and compared to amiloride and benzamilas positive controls.

Pharmacological Assays of Absorption

(1) Apical Disappearance Assay

Bronchial cells (dog, human, sheep, or rodent cells) are seeded at adensity of 0.25×10⁶/cm² on a porous Transwell-Col collagen-coatedmembrane with a growth area of 1.13 cm² grown at an air-liquid interfacein hormonally defined media that promotes a polarized epithelium. From12 to 20 days after development of an air-liquid interface (ALI) thecultures are expected to be >90% ciliated, and mucins will accumulate onthe cells. To ensure the integrity of primary airway epithelial cellpreparations, the transepithelial resistance (Rt) and transepithelialpotential differences (PD), which are indicators of the integrity ofpolarized nature of the culture, are measured. Human cell systems arepreferred for studies of rates of absorption from apical surfaces. Thedisappearance assay is conducted under conditions that mimic the “thin”films in vivo (˜25 μl) and is initiated by adding experimental sodiumchannel blockers or positive controls (amiloride, benzamil, phenamil) tothe apical surface at an initial concentration of 10 μM. A series ofsamples (5 μl volume per sample) is collected at various time points,including 0, 5, 20, 40, 90 and 240 minutes. Concentrations aredetermined by measuring intrinsic fluorescence of each sodium channelblocker using a Fluorocount Microplate Flourometer or HPLC. Quantitativeanalysis employs a standard curve generated from authentic referencestandard materials of known concentration and purity. Data analysis ofthe rate of disappearance is performed using nonlinear regression, onephase exponential decay (Prism V 3.0).

2. Confocal Microscopy Assay of Amiloride Congener Uptake

Virtually all amiloride-like molecules fluoresce in the ultravioletrange. This property of these molecules may be used to directly measurecellular update using x-z confocal microscopy. Equimolar concentrationsof experimental compounds and positive controls including amiloride andcompounds that demonstrate rapid uptake into the cellular compartment(benzamil and phenamil) are placed on the apical surface of airwaycultures on the stage of the confocal microscope. Serial x-z images areobtained with time and the magnitude of fluorescence accumulating in thecellular compartment is quantitated and plotted as a change influorescence versus time.

3. In Vitro Assays of Compound Metabolism

Airway epithelial cells have the capacity to metabolize drugs during theprocess of transepithelial absorption. Further, although less likely, itis possible that drugs can be metabolized on airway epithelial surfacesby specific ectoenzyme activities. Perhaps more likely as anecto-surface event, compounds may be metabolized by the infectedsecretions that occupy the airway lumens of patients with lung disease,e.g. cystic fibrosis. Thus, a series of assays is performed tocharacterize the compound metabolism that results from the interactionof test compounds with human airway epithelia and/or human airwayepithelial lumenal products.

In the first series of assays, the interaction of test compounds in KBRas an “ASL” stimulant are applied to the apical surface of human airwayepithelial cells grown in the T-Col insert system. For most compounds,metabolism (generation of new species) is tested for using highperformance liquid chromatography (HPLC) to resolve chemical species andthe endogenous fluorescence properties of these compounds to estimatethe relative quantities of test compound and novel metabolites. For atypical assay, a test solution (25 μl KBR, containing 10 μM testcompound) is placed on the epithelial lumenal surface. Sequential 5 to10 μl samples are obtained from the lumenal and serosal compartments forHPLC analysis of (1) the mass of test compound permeating from thelumenal to serosal bath and (2) the potential formation of metabolitesfrom the parent compound. In instances where the fluorescence propertiesof the test molecule are not adequate for such characterizations,radiolabeled compounds are used for these assays. From the HPLC data,the rate of disappearance and/or formation of novel metabolite compoundson the lumenal surface and the appearance of test compound and/or novelmetabolite in the basolateral solution is quantitated. The data relatingthe chromatographic mobility of potential novel metabolites withreference to the parent compound are also quantitated.

To analyze the potential metabolism of test compounds by CF sputum, a“representative” mixture of expectorated CF sputum obtained from 10 CFpatients (under IRB approval) has been collected. The sputum has been besolubilized in a 1:5 mixture of KBR solution with vigorous vortexing,following which the mixture was split into a “neat” sputum aliquot andan aliquot subjected to ultracentrifugation so that a “supernatant”aliquot was obtained (neat=cellular; supernatant=liquid phase). Typicalstudies of compound metabolism by CF sputum involve the addition ofknown masses of test compound to “neat” CF sputum and aliquots of CFsputum “supernatant” incubated at 37° C., followed by sequentialsampling of aliquots from each sputum type for characterization ofcompound stability/metabolism by HPLC analysis as described above. Asabove, analysis of compound disappearance, rates of formation of novelmetabolities, and HPLC mobilities of novel metabolites are thenperformed.

4. Pharmacological Effects and Mechanism of Action of the Drug inAnimals

The effect of compounds for enhancing mucociliary clearance (MCC) can bemeasured using an in vivo model described by Sabater et al., Journal ofApplied Physiology, 1999, pp. 2191-2196, incorporated herein byreference.

EXAMPLES

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

Preparation of Sodium Channel Blockers

Materials and methods. All reagents and solvents were purchased fromAldrich Chemical Corp. and used without further purification. NMRspectra were obtained on either a Bruker WM 360 (¹H NMR at 360 MHz and¹³C NMR at 90 MHz) or a Bruker AC 300 (¹H NMR at 300 MHz and ¹³C NMR at75 MHz). Flash chromatography was performed on a Flash Elute™ systemfrom Elution Solution (PO Box 5147, Charlottesville, Va. 22905) chargedwith a 90 g silica gel cartridge (40M FSO-0110-040155, 32-63 μm) at 20psi (N₂). GC-analysis was performed on a Shimadzu GC-17 equipped with aHeliflex Capillary Column (Alltech); Phase: AT-1, Length: 10 meters, ID:0.53 mm, Film: 0.25 micrometers. GC Parameters: Injector at 320° C.,Detector at 320° C., FID gas flow: H₂ at 40 ml/min., Air at 400 ml/min.Carrier gas: Split Ratio 16:1, N₂ flow at 15 ml/min., N₂ velocity at 18cm/sec. The temperature program is 70° C. for 0-3 min, 70-300° C. from3-10 min, 300° C. from 10-15 mm.

HPLC analysis was performed on a Gilson 322 Pump, detector UV is 156 at360 nm, equipped with a Microsorb MV C8 column, 100 A, 25 cm. Mobilephase: A=acetonitrile with 0.1% TFA, B=water with 0.1% TFA. Gradientprogram: 95:5 B:A for 1 min, then to 20:80 B:A over 7 min, then to 100%A over 1 min, followed by washout with 100% A for 11 min, flow rate: 1ml/min.

Example 14-{4-[N-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-N-(2-aminoethyl)benzamidehydrochloride (11698)

4-(4-Aminobutyl)benzoic acid (30)

A solution of sodium hydroxide (0.69 g, 17.37 mmol) in water (30 mL) wasadded to a solution of 24 (1.2 g, 5.79 mmol) in methanol (30 mL) andstirred at room temperature for 48 h. Then the solvent was removed underreduced pressure. Water (20 mL) was added and pH was adjusted to 7 withHCl. The white solid precipitate was filtered off, washed with water anddried in vacuum. The crude product 30 (1.39 g) was obtained as a whitesolid and used for the next step without further purification.

4-(4-Benzyloxycarbonylaminobutyl)benzoic acid (31)

Sodium hydrogencarbonate (0.95 g, 11.32 mmol) was added into asuspension of 30 in THF (120 mL), followed by water (10 mL), affording aclear solution. Benzyl chloroformate (1.21 mL, 8.49 mmol) was then addedinto the reaction mixture at 0° C. The reaction mixture was then stirredat room temperature overnight. After that, the solvent was removed underreduced pressure. Ethyl acetate (70 mL) was added to the residue and thesolution was washed with 2N HCl (2×30 mL) and water (2×50 mL), thendried in vacuum. 1.82 g (98%) of 31 was obtained as a white solid. ¹HNMR (300 MHz, DMSO-d₆) δ 1.11 (m, 2H), 1.28 (m, 2H), 2.33 (m, 2H), 3.02(m, 2H), 5.01 (m, 2H), 7.15 (m, 7H), 7.93 (d 2H).

4-[4-(2-tert-Butoxycarbonylaminoethylcarbamoyl)phenyl]butyl}carbamicacid benzyl ester (32)

N,N′-Dicyclohexylcarbodiimide (DCC) (0.69 g, 3.36 mmol) was added to acold (0° C.) methylene chloride solution of 31 (1 g, 3.05 mmol) and1-hydroxybenzotriazole (HOBt) (0.41 g, 3.05 mmol) under a nitrogenatmosphere. The reaction mixture was then stirred at room temperatureovernight. A white precipitate was formed. The solvent was removed underreduced pressure and the residue was separated by Flash™ (BIOTAGE, Inc)(90 g silica gel cartridge 40M, 3:2 methylene chloride/ethyl acetate) toprovide 32 (0.9 g, 63%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ1.70 (m, 11H), 1.94 (m, 2H), 3.23 (m, 2H), 3.72 (m, 2H), 3.82 (m, 2H),4.08 (m, 2H), 6.19 (s, 2H), 8.61 (m, 1H), 9.04 (m, 7H), 9.61 (d, 2H).

{2-[4-(4-Aminobutyl)benzoylamino]ethyl}carbamic acid tert-butyl ester(33)

A suspension of 32 (0.9 g, 1.92 mmol) with 10% palladium on carbon (0.30g, wet) in methanol (50 mL) was stirred for 2 h at room temperatureunder atmospheric pressure of hydrogen. The mixture was then filteredthrough a silica gel pad. The solvent was evaporated and the residue waspurified by Flash™ (BIOTAGE, Inc) (90 g silica gel cartridge 40M,6:1:0.1 chloroform/ethanol/concentrated ammonium hydroxide) to provide33 (0.405 g, 63%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.08 (m,2H), 1.37 (s, 9H), 1.58 (m, 2H), 2.52 (m, 2H), 3.28 (m, 2H), 6.91 (m,1H), 7.27 (d, 2H), 7.74 (d, 2H), 8.39 (m, 1H).

[2-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-benzoylamino)ethyl]carbamicacid tert-butyl ester (34)

1-(3,5-Diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (0.32g, 0.82 mmol) and triethylamine (0.27 mL, 1.64 mmol) were sequentiallyadded into a solution of 33 (0.39 g, 0.82 mmol) in 5 mL of methanol. Thereaction mixture was stirred in the boiling solvent for 2 h. The solventwas evaporated and the residue was separated by Flash™ (BIOTAGE, Inc)(90 g silica gel cartridge 40M, 6:1:0.1 chloroform/ethanol/concentratedammonium hydroxide) to provide 34 (0.39 g, 62%) as a yellow solid. ¹HNMR (300 MHz, DMSO-d₆) δ 1.07 (t, 2H), 1.38 (s, 9H), 1.52 (m, 2H), 1.65(m, 2H), 2.66 (m, 2H), 3.10 (m, 2H), 3.28 (m, 2H), 4.39 (m, 1H), 6.65(br s, 1H), 6.94 (m, 1H), 7.28 (m, 2H), 7.75 (d, 2H), 8.49 (m, 1H). m/z(APCI)=548 [C₂₄H₃₄ClN₉O₄+H]⁺.

N-(2-Aminoethyl)-4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-benzamidehydrochloride (35 (11698))

The solution of 34 (0.104 g, 0.19 mmol) in a mixture of methanol/HCl(1:1, 8 mL) was stirred at room temperature for 0.5 h; then the solventwas completely evaporated, affording 0.099 g (100%) of 35 as a yellowsolid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.60 (m, 4H), 2.69 (m, 2H), 2.98 (m,2H), 3.33 (m, 2H), 3.52 (m, 2H), 7.32 (d, 2H), 7.90 (d, 2H), 8.23 (br s,2H), 8.82 (m, 1H), 8.90 (br s, 1H), 9.02 (br s, 1H), 9.37 (m, 1H), 10.57(s, 1H). m/z (APCI)=448 [C₁₉H₂₆ClN₉O₂+H]⁺.

Example 24-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-N-(2-guanidinoethyl)benzamidehydrochloride (11834)

4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-N-[2-N″,N′″-di-(butyloxycarbonylguanidino-ethyl]benzamide(36)

Triethylamine (0.34 mL, 2.44 mmol) was added into a suspension of 35 inmethanol (25 mL). The reaction mixture was stirred at room temperaturefor 20 min; at which time the suspension became a clear solution.N,N′-di-(tert-butoxycarbonyl)-N″-trifluoromethansulfonylguanidine(Goodman's reagent) (0.193 g, 0.489 mmol) was added into the reaction.The reaction mixture was stirred at room temperature for additional 6 h,after that the solvent was removed under reduced pressure and theresidue was purified by Flash™ (BIOTAGE, Inc) (90 g silica gel cartridge40M, 6:1:0.1 chloroform/ethanol/concentrated ammonium hydroxide) toprovide 36 (0.18 g, 80%) as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ1.40 (s, 9H), 1.47 (s, 9H), 1.52 (m, 2H), 1.65 (m, 2H), 2.68 (m, 2H),3.18 (br s, 2H), 3.40 (m, 2H), 3.49 (m, 2H), 6.77 (br s, 2H), 7.30 (d,2H), 7.75 (d, 2H), 8.50 (br s, 2H).

4-{4-[N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl)-N-(2-guanidino-ethyl)benzamidehydrochloride (37 (11834))

The solution of 36 (0.155 g, 0.22 mmol) in a mixture of methanol/HCl(1:1, 4 mL) was stirred at room temperature for 2 h, then the solventwas evaporated and the residue dried in vacuum to provide 0.126 g (100%)of 37 as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.54 (m, 2H), 1.65(m, 2H), 2.68 (m, 2H), 3.35 (br.s, 4H), 7.32 (d, 2H), 7.90 (d, 2H), 8.79(m, 1H), 8.92 (br s, 1H), 8.90 (br s, 1H), 9.02 (br s, 1H), 9.37 (m,1H), 10.57 (s, 1H). m/z (APCI)=490 [C₂₀H₂₈ClN₁₁O₂+H]⁺.

Example 3N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-(3-guanidino-2-hydroxypropoxy)phenyl]butyl}guanidinehydrochloride (11975)

The synthesis of [4-(4-allyloxyphenyl)butyl]carbamic acid benzyl ester(38) was described in the previously provided experimental details (ascompound 30).

[4-(4-Oxiranylmethoxyphenyl)butyl]carbamic acid benzyl ester (39)

3-Chloro-peroxybenzoic acid (2.46 g, 14.25 mmol) was added into amethylene chloride solution (100 mL) of 38 (1.86 g, 5.48 mmol), and thereaction was stirred at room temperature overnight. After that, thesolvent was removed under reduced pressure and the residue was purifiedby flash chromatography (silica gel, 8:1:1 methylenechloride/hexane/ethyl acetate). To eliminate the admixture of benzoicacid the methylene chloride solution of the product was sequentiallywashed with a saturated aqueous solution of sodium hydrogen carbonateand water, then dried over anhydrous sodium sulfate and evaporated toprovide 1.4 g (72%) of 39 as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ1.57 (m, 4H), 2.56 (m, 2H), 2.78 (m, 1H), 2.91 (m, 1H), 3.21 (m, 2H),3.36 (m, 1H), 3.97 (m, 1H), 4.19 (m, 1H), 5.08 (s, 2H), 6.82 (d, 2H),7.06 (d, 2H), 7.72 (s, 5H).

{4-[4-(3-Amino-2-hydroxypropoxy)phenyl]butyl}carbamic acid benzyl ester(40)

An ethanol solution of 39 (0.86 g, 2.42 mmol) was saturated with ammoniaand stirred overnight at room temperature. The solvent was -evaporatedand the residue was purified by Flash™ (BIOTAGE, Inc) (90 g silica gelcartridge 40M, 6:1:0.1 chloroform/ethanol/concentrated ammoniumhydroxide) to provide 40 (0.75 g, 87%) as a white solid. ¹H NMR (300MHz, DMSO-d₆) δ 1.40 (m, 2H), 1.51 (m, 2H), 2.50 (m, 2H), 2.58 (m, 1H),2.68 (m, 1H), 3.00 (m, 2H), 3.69 (m, 1H), 3.80 (m, 1H), 3.90 (m, 1H),5.08 (s, 2H), 6.82 (d, 2H), 7.06 (d, 2H), 7.35 (br s, 5H).

{4-[4-(3-tert-Butoxycarbonylamino-2-hydroxpropoxy)-phenyl]butyl}carbamicacid tert-butyl ester (41)

The compound 41 was prepared in a similar manner to the synthesis ofcompound 25, starting from compound 40 (0.75 g, 2.03 mmol). It waspurified by Flash™ (BIOTAGE, Inc) (90 g silica gel cartridge 40M, 2:1hexane/ethyl acetate) as a white solid (0.8 g, 83%). ¹H NMR (300 MHz,CDCl₃) δ 1.45 (s, 9H), 1.57 (m, 4H), 2.58 (m, 2H), 3.20 (m, 2H), 3.31(m, 2H), 3.94 (m, 2H), 4.10 (m, 1H), 4.73 (br s, 1H), 5.00 (br s, 1H),5.10 (s, 2H), 6.81 (d, 2H), 7.06 (d, 2H), 7.35 (br s, 5H).

{3-[4-(4-Aminobutyl)phenoxy]-2-hydroxypropyl}carbamic acid tert-butylester (42)

A suspension of 41 (0.8 g, 1.69 mmol) with 10% palladium on carbon (0.40g, wet) in methanol (30 mL) was stirred for 3 h at room temperatureunder atmospheric pressure of hydrogen. The mixture was then filteredthrough a silica gel pad; the solvent was evaporated to provide 42(0.705 g, 99%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 1.45 (s, 9H),1.55 (m, 4H), 2.58 (m, 4H), 2.71 (m, 2H), 3.29 (m, 1H), 3.45 (m, 2H),3.92 (m, 2H), 4.10 (br s, 1H), 5.10 (br s, 1H), 6.81 (d, 2H), 7.08 (d,2H).

[3-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)-2-hydroxypropyl]carbamicacid tert-butyl ester (43)

Compound 43 was prepared in a similar manner to the synthesis ofcompound 29, starting from compound 42 (0.46 g, 1.36 mmol). It waspurified by Flash™ (BIOTAGE, Inc) (90 g silica gel cartridge 40M,6:1:0.1 chloroform/ethanol/concentrated ammonium hydroxide) as a yellowsolid (0.37 g, 57%). ¹H NMR (300 MHz, DMSO-d₆) δ 1.38 (s, 9H), 1.58 (brs, 4H), 2.55 (m, 2H), 3.00 (m, 2H), 3.08 (m, 2H), 3.33 (m, 2H), 3.82 (m,3H), 5.13 (br s, 1H), 6.85 (d, 2H), 7.10 (d, 2H), 7.46 (br s, 2H).

N-{4-[4-(4-Amino-2-hydroxypropoxy)phenyl]butyl}-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine(44)

The free base of compound 44 was prepared in a similar manner to thesynthesis of the compound 28, starting from the compound 43 (0.18 g,0.33 mmol) and purified by Flash™ (BIOTAGE, Inc) (90 g silica gelcartridge 40M, 2:1:0.1 chloroform/ethanol/concentrated ammoniumhydroxide) to afford the product free base as a yellow solid. It wasthen treated with 3% HCl. The solvent was evaporated, and the residuewas dried in vacuum to afford 0.125 g (73%) of the compound 44. ¹H NMR(300 MHz, DMSO-d₆) δ 1.58 (br s, 4H), 2.55 (m, 2H), 2.84 (br s, 1H),3.03 (br s, 1H), 3.34 (br s, 2H), 3.94 (m, 2H), 5.13 (br s, 1H), 6.85(d, 2H), 7.13 (d, 2H), 8.13 (br s, 2H), 8.90 (br s, 1H), 9.00 (br s,1H), 9.34 (br s, 1H), 10.56 (s, 1H).

N-(3,5-Diamino-6-chloro-pyrazine-2-carbonyl)-N′-{4-[4-(3-(N″,N′″-di-tert-butyl-oxycarbonyl)-guanidino)-2-hydroxy-propoxy)phenyl]-butyl}-guanidine(45)

Triethylamine (0.30 mL, 2.14 mmol) was added into a solution of 44 inmethanol (10 mL) followed by the addition ofN,N′-di-(tert-butoxycarbonyl)-N″-trifluoromethansulfonyl-guanidine(Goodman's reagent) (0.169 g, 0.4295 mmol). The reaction mixture wasstirred at room temperature for 2 h. After that the solvent was removedunder reduced pressure and the residue was purified by Flash™ (BIOTAGE,Inc) (90 g silica gel cartridge 40M, 6:1:0.1chloroform/ethanol/concentrated ammonium hydroxide) to provide 45 (0.154g, 78%) as a yellow solid. ¹H NMR (300 MHz, CD₃OD) δ 1.47 (s, 9H), 1.52(s, 9H), 1.66 (br s, 4H), 2.58 (m, 2H), 3.23 (br s, 2H), 3.48 (m, 1H),3.70 (m, 1H), 3.95 (m, 2H), 4.09 (m, 1H), 6.88 (d, 2H), 7.10 (d, 2H).

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-(3-guanidino-2-hydroxy-propoxy)phenyl]butyl}guanidinehydrochloride (46)

A solution of 45 (0.134 g, 0.19 mmol) in concentrated hydrochloric acidwas stirred at room temperature for 0.5 h. The solvent was thenevaporated and the residue dried in vacuum to provide 0.108 g (99%) of46 as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.60 (br s, 4H), 2.52(br s, 1H), 3.28 (m, 1H), 3.36 (m, 2H), 4.91(s, 2H), 6.88 (d, 2H), 7.12(d, 2H), 7.79 (m, 1H), 8.92 (br s, 1H), 9.03 (br s, 1H), 9.37 (m, 1H),10.57 (s, 1H). m/z (APCI)=493 [C₂₀H₂₉ClN₁₀O₃+H]⁺.

Example 4N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[4-(4-bis(methanesulfonyl)aminophenyl)butyl]guanidine(10316)

[4-(4-Nitrophenyl)but-3-ynyl]carbamic acid tert-butyl ester (51)

To a mixture of anhydrous THF and triethylamine (20 mL, 1/1) weresequentially added 1-iodo-4-nitrobenzene (2.0 g, 8.032 mmol) and copper(1) iodide (0.31 g, 1.606 mmol). The mixture was stirred at roomtemperature for 15 min. The flask was evacuated and re-filled with argonfour times to ensure no oxygen remained. The catalyst,dichlorobis(triphenylphosphine)-palladium(II) (0.56 g, 0.803 mmol) wasadded into the mixture under argon protection, followed by dropwiseaddition of but-3-ynyl-carbamic acid tert-butyl ester (1.62 g, 9.638mmol). The newly formed reaction mixture was further stirred at roomtemperature overnight. The solid in the reaction mixture was vacuumfiltered. The filtrate was concentrated. The residue was re-dissolved inmethylene chloride and purified by column chromatography, eluting with amixture of ethyl acetate (0-10%) and hexanes (100-90%) to afford 2.08 g(89%) of the product 51 as an orange solid. ¹H NMR (CDCl₃) δ 1.46 (s,9H), 2.66 (t, J=6.5 Hz, 2H), 3.36-3.43 (m, 2H), 4.86 (br s, 1H), 7.53(d, J=8.7 Hz, 2H), 8.16 (d, J=8.7 Hz, 2H). m/z (APCI)=291[C₁₅H₁₈N₂O₄+H]⁺, 191 [C₁₅H₁₈N₂O₄−Boc+H]⁺.

[4-(4-Aminophenyl)butyl]carbamic acid tert-butyl ester (52)

To a solution of compound 51 (2.01 g, 6.923 mmol) in ethanol (50 mL) wasadded 10% palladium on carbon (737 mg, wet) in one portion under argonprotection. The flask was evacuated and re-filled with argon three times(to remove oxygen), and the mixture stirred at room temperatureovernight under one atmosphere of hydrogen. The reaction system was thenpurged with nitrogen, and the catalyst was vacuum filtered and washedwith ethanol (2×5 mL). The filtrate and washings were combined andconcentrated under reduced pressure. The residue was chromatographedover silica gel, eluting with a mixture of ethyl acetate (0-25%) andhexanes (100-75%), to afford 1.75 g (96%) of the product 52 as acolorless viscous oil. ¹H NMR (CDCl₃) δ 1.44 (s, 9H), 1.47-1.64 (m, 4H),2.50 (t, J=7.1 Hz, 2H), 3.08-3.14 (m, 2H), 3.57 (br s, 2H), 4.55 (br s,1H), 6.62 (d, J=8.2 Hz, 2H), 6.95 (d, J=8.2 Hz, 2H). m/z (APCI)=265[C₁₅H₂₄N₂O₂+H]⁺.

4-{4-[Bis(methanesulfonyl)amino]phenyl}butylcarbamic acid tert-butylester (53)

Compound 52 (0.16 g, 0.605 mmol) was dissolved in anhydrous THF (5 mL).To the clear solution were sequentially added triethylamine (0.18 mL,1.21 mmol) and 4-dimethylaminopyridine (15 mg, 0.121 mmol). The mixturewas cooled to about −10° C. for 15 min by a methanol-ice bath. To thecold solution was slowly added methanesulfonyl chloride (51 μL). Thesolution was further stirred at the temperature (about −10° C.) for anadditional 30 min, then allowed to slowly warm up to room temperature byremoving the cooling bath. The reaction mixture was concentrated underreduced pressure. The residue was chromatographed over silica gel,eluting with a mixture of ethyl acetate (0-35%) and hexanes (100-65%),to afford 0.212 g (83%) of the product 53 as a white solid. ¹H NMR(CDCl₃) δ 1.44 (s, 9H), 1.52-1.70 (m, 4H), 2.68 (t, J=7.4 Hz, 2H),3.13-3.18 (m, 2H), 3.40 (s, 6H), 4.53 (br s, 1H), 7.27 (s, 4H). m/z(APCI)=321 [C₁₇H₂₈N₂O₆S₂−Boc+H]⁺.

4-[4-Bis(methanesulfonyl)amino)phenyl]butylamine (54)

A solution of compound 53 (0.21 g, 0.499 mmol) dissolved in methylenechloride (10 mL) was treated with trifluoroacetic acid (1 mL) at roomtemperature for 2 hours, then concentrated under vacuum. The residue wastaken into methanol (2 mL), and concentrated again under reducedpressure. The procedure was repeated three times to ensure no residualtrifluoroacetic acid remained. The product was completely dried undervacuum, and directly used for the next reaction without furtherpurification. 0.196 g (100%) of the compound 54 was obtained as acolorless viscous oil. ¹H NMR (DMSO-d₆) δ 1.50-1.72 (m, 4H), 2.65 (t,J=7.4 Hz, 2H), 2.84-2.89 (m, 2H), 3.51 (s, 6H), 7.33 (d, J=8.3 Hz, 2H),7.42 (d, J=8.3 Hz, 2H). m/z (APCI)=321 [C₁₂H₂₀N₂O₄S₂+H]⁺.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-[4-(4-bis(methanesulfonyl)-aminophenyl)butyl]guanidine(55, 10316)

Compound 54 (0.095 g, 0.296 mmol) was mixed with ethanol (5 mL). Themixture was heated at 65° C. for 15 min to achieve complete dissolution.To the clear solution were sequentially added diisopropylethylamine(0.26 mL, 1.48 mmol) and1-(3,5-diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (0.127g, 0.326 mmol). The mixture was heated at the same temperature for anadditional 1.5 hours, and subsequently concentrated under vacuum. Theresidue was chromatographed over silica gel, eluting with a mixture ofconcentrated ammonium hydroxide (0-1%), methanol (0-10%), and methylenechloride (100-89%), to afford 0.113 g (72%) of the product 55 as a lightyellow solid. mp 174-176° C. (decomposed). ¹H NMR (DMSO-d₆) δ 1.48-1.68(m, 4H), 2.64-2.69 (m, 2H), 3.12-3.25 (m, 2H), 3.51 (s, 6H), 6.65-6.78(br s, 3H), 7.31 (d, J=8.2 Hz, 2H), 7.41 (d, J=8.2 Hz, 2H), 9.05 (br s,2H). m/z (APCI)=533 [C₁₈H₂₅ClN₈O₅S₂+H]⁺.

Example 5N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-{4-[4-(2-chlorotrimethylammonium)ethoxyphenyl]butyl}guanidinehydrochloride (11223)

{4-[4-(2-Dimethylaminoethoxy)phenyl]butyl}carbamic acid benzyl ester(62)

A mixture of [4-(4-hydroxyphenyl)butyl]carbamic acid benzyl ester (1.5g, 5 mmol), 2-dimethylaminochloroethane hydrochloride (1.4 g, 10 mmol),potassium carbonate (2.76 g, 20 mmol) and 18-crown-6 ether (154 mg, 0.58mmol) was stirred at 80° C. (oil bath) for 18 h. After this time, thesolvent was removed under reduced pressure and the residue was purifiedby Flash™ (BIOTAGE, Inc) (90 g silica gel cartridge 40M, 10:1:0.1chloroform/methanol/concentrated ammonium hydroxide) to provide 62 (1.1g, 61%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 1.56 (m, 4H), 2.32(s, 6H), 2.56 (m, 2H), 2.71 (t, 2H), 3.18 (m, 2H), 4.04 (t, 2H), 4.78(br s, 1H), 5.08 (s, 2H), 6.83 (d, 2H), 7.06 (d, 2H), 7.35 (m, 5H).

4-[4-(2-Dimethylaminoethoxy)phenyl]butylamine (63)

The protected amine 62 (0.552 g, 1.5 mmol) was stirred with 10%palladium on carbon (0.127 g, wet) in methanol (50 mL) at roomtemperature for 3.5 h under hydrogen (1 atm). After this time, thecatalyst was filtered off and the solvent was removed under reducedpressure to give the free amine 63 (0.27 g, 77%) as a white powder. ¹HNMR (300 MHz, CD₃OD) δ 1.55 (m, 4H), 2.32 (s, 6H), 2.54 (m, 2H), 2.75(m, 2H), 4.02 (m, 2H), 6.78 (d, 2H), 7.08 (d, 2H).

1-tert-Butyloxycarbonyl-3-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methyl-isothiourea(64)

4-Dimethylaminopyridine (87 mg, 0.7 mmol) was added to a stirringsolution of di-tert-butyl dicarbonate (0.8 g, 3.6 mmol) and1-(3,5-diamino-6-chloropyrazinoyl)-2-methylisothiourea hydriodide (1.14g, 0.5 mmol) in THF/triethylamine (62 mL, 30/1). The reaction mixturewas then stirred at room temperature for 48 h. After this time, thesolvent was removed under reduced pressure. The residue was purified byflash chromatography (silica gel, 1:1 ethyl acetate/hexanes) to give theprotected isothiourea 64 (0.34 g, 32%) as a yellow powder. ¹H NMR (300MHz, DMSO-d₆) δ 1.51 (s, 9H), 2.30 (s, 3H), 7.40 (br s, 4H).

N-tert-Butyloxycarbonyl-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N″-{4-[4-(2-dimethylaminoethoxy)phenyl]butyl}guanidine(65)

A suspension of compound 63 (0.22 g, 0.93 mmol) and 64 (0.33 g, 0.92mmol) in THF/triethylamine (11 mL, 10/1) was stirred at room temperaturefor 48 h. After this time, a clear solution was formed. The solvent wasremoved under reduced pressure and the residue was purified by flashchromatography (silica gel, 10:1:0.1 chloroform/methanol/concentratedammonium hydroxide) to provide the guanidine 65 (0.3 g, 60%) as a yellowsolid. ¹H NMR (300 MHz, DMSO-d₆) δ 1.42 (s, 9H), 1.55 (m, 4H), 2.19 (s,6H), 2.58 (m, 4H), 3.99 (m, 2H), 6.83 (d, 2H), 7.12 (d, 2H), 7.40 (br s,2H), 9.02 (m, 2H).

N-tert-Butyloxycarbonyl-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N″-{4-[4-(2-iodotrimethylammoniumethoxy)phenyl]butyl}guanidine(66)

Iodomethane (30 μL, 0.49 mmol) was added to a suspension of 65 (0.29 g,0.52 mmol) in THF (50 mL). The mixture was stirred at room temperatureovernight. After this time, additional THF (7 mL) was added and stirringwas continued for 2 d to give a clear solution. The solvent from theresulting solution was removed under reduced pressure. The residue waswashed with THF (2×5 mL) and dried to afford the salt 66 (0.22 g, 32%)as a yellow powder. ¹H NMR (300 MHz, DMSO-d₆) δ 1.42 (s, 9H), 1.56 (m,4H), 2.58 (m, 2H), 3.15 (s, 9H), 3.75 (m, 2H), 4.42 (m, 2H), 6.92 (d,2H), 7.18 (d, 2H), 7.36 (br s, 2H), 9.02 (m, 1H).

[2-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)ethyl]-trimethylammoniumchloride (67, 11223)

Trifluoroacetic acid (2 mL) was added to the protected guanidine 66(0.092 g, 0.13 mmol). The reaction mixture was stirred at roomtemperature for 15 min. The solvent was removed under reduced pressureand the residue was washed with ethyl acetate (2×1 mL) and dried invacuum. The obtained dry solid was treated with an aqueous solution ofammonium hydroxide (15%, 1 mL). The formed precipitate was collected bycentrifugation and washed with cold water (1 mL). The remaining solidwas dissolved in 10% hydrochloric acid, and the solvent was then removedunder reduced pressure. The resulting yellow solid was dried in vacuumto give compound 67 (0.055 g, 82%). ¹H NMR (300 MHz, CD₃OD) δ 1.68 (brs, 4H), 2.65 (m, 2H), 3.35 (m, 2H), 3.83 (m, 2H), 4.46 (m, 2H), 4.95 (s,9H), 6.96 (d, 2H), 7.18 (d, 2H), 9.25 (br s, 1H). m/z (APCI)=499[C₂₁H₃₂Cl₂N₈O₂+H]⁺.

Sodium Channel Blocking Activity

The compounds shown in the Tables below were tested for potency incanine bronchial epithelia using the in vitro assay described above.Amiloride was also tested in this assay as a positive control. Theresults for the compounds of the present invention are reported asfold-enhancement values relative to amiloride.

Example 6

X═ Y═ Fold Amiloride* —NHCO₂+ —CO₂CH₃ 29.4 ± 10 (5)   | | | NH₂ —CO₂H17.2 ± 5.8 (4)  | | | NH₂ —CO₂CH₃ 21.1 ± 14.1 (2) | | | | |NH₂ —CONH₂*Relative potency for Amiloride = 665 nM. **Relative potency for CF552 =100 using IC₅₀ from 552 in same run. ***3^(rd) Wash (a) Old Database (b)NA = Not Available (c) 1 of 5 is high outlier (252)

Example 7

X═ Fold Amiloride* N(CH₃)₂ 25.3 ± 2.3 (3) N⊕(CH₃)₃ 39.4 ± 0.3 (2)*Relative potency for Amiloride = 665 nM. **Relative potency for CF552 =100 using IC₅₀ from 552 in same run. ***3^(rd) Wash a Guanidinine isAcylated

Example 8

  R═\Z═

  NH(C═O)CH₂CH₂R NH₂ Xamiloride 86.2 ± 30.1 170   R═\Z═

Xamiloride 182.3 ± 106.3 (12) 139.7 ± 62.4

References. 1. Rappoport, D. A.; Hassid, Z.; J. Amer. Chem. Soc., 1951,73, 5524-5525, incorporated herein by reference.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1. A method of increasing hydration of a mucosal surface in a subject,comprising topically administering to a mucosal surface of a subject apyrazinoylguanidine compound represented by formula (I):

where X is hydrogen, halogen, trifluoromethyl, lower alkyl,unsubstituted or substituted phenyl, lower alkyl-thio, phenyl-loweralkyl-thio, lower alkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl; Y ishydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio, halogen,lower alkyl, unsubstituted or substituted mononuclear aryl, or —N(R²)₂;R¹ is hydrogen or lower alkyl; each R² is, independently, —R⁷,—(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-Z_(g)-R⁷, —(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

wherein when two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect toeach other the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane; R³ and R⁴ are each,independently, hydrogen, a group represented by formula (A), loweralkyl, hydroxy lower alkyl, phenyl, phenyl-lower alkyl,(halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, with the proviso that at least one of R³ and R⁴ is a grouprepresented by formula (A):

wherein  each R^(L) is, independently, —R⁷, —(CH₂)_(n)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

wherein when two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect toeach other the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane; each o is, independently,an integer from 0 to 10; each p is an integer from 0 to 10; with theproviso that the sum of o and p in each contiguous chain is from 1 to10; each x is, independently, O, NR¹⁰, C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰,or represents a single bond; each R⁵ is, independently,—(CH₂)_(n)—NR¹²R¹², —O—(CH₂)_(m)—NR¹²R¹², —O—(CH₂)_(n)—NR¹²R¹²,—O—(CH₂)_(m)-(Z)_(g)-R¹², —(CH₂)_(n)NR¹¹R¹¹, —O—(CH₂)_(m)NR¹¹R¹¹,—(CH₂)_(n)—N^(⊕)—(R¹¹)₃, —O—(CH₂)_(m)—N^(⊕)—(R¹¹)₃,—(CH₂)_(n)-(Z)_(g)-(CH₂)_(m)—NR¹⁰R¹⁰,—O—(CH₂)_(m)-(Z)_(g)-(CH₂)_(m)—NR¹⁰R¹⁰, —(CH₂CH₂O)_(m)—CH₂CH₂NR¹²R¹²,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR¹²R¹², —(CH₂)_(n)—(C═O)NR¹²R¹²,—O—(CH₂)_(m)—(C═O)NR¹²R¹², —O—(CH₂)_(m)—(CHOR⁸)_(m)CH₂NR¹⁰-(Z)_(g)-R¹⁰,—(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹⁰-(Z)_(g)-R¹⁰,—(CH₂)_(n)NR¹⁰—O(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹⁰-(Z)_(g)-R¹⁰,—O(CH₂)_(m)—NR¹⁰—(CH₂)_(m)—(CHOR⁸)_(n)CH₂NR¹⁰-(Z)_(g)-R¹⁰,-(Het)-(CH₂)_(m)—OR⁸, -(Het)-(CH₂)_(m)—NR⁷R¹⁰,-(Het)-(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, -(Het)-(CH₂CH₂O)_(m)—R⁸,-(Het)-(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, -(Het)-(CH₂)_(m)—C(═O)NR⁷R¹⁰,-(Het)-(CH₂)_(m)-(Z)_(g)-R⁷,-(Het)-(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,-(Het)-(CH₂)_(m)—CO₂R⁷, -(Het)-(CH₂)_(m)—NR¹²R¹²,-(Het)-(CH₂)_(n)—NR¹²R¹², -(Het)-(CH₂)_(m)-(Z)_(g)-R¹²,-(Het)-(CH₂)_(m)NR¹¹R¹¹, -(Het)-(CH₂)_(m)—N^(⊕)—(R¹¹)₃,-(Het)-(CH₂)_(m)-(Z)_(g)-(CH₂)_(m)—NR¹⁰R¹⁰,-(Het)-(CH₂CH₂O)_(m)—CH₂CH₂NR¹²R¹², -(Het)-(CH₂)_(m)—(C═O)NR¹²R¹²,-(Het)-(CH₂)_(m)—(CHOR⁸)_(m)CH₂NR¹⁰-(Z)_(g)-R¹⁰,-(Het)-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)—(CHOR⁸)_(n)CH₂NR¹⁰-(Z)_(g)-R¹⁰, whereinwhen two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect to eachother the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with the proviso that at least two—CH₂OR⁸ are located adjacent to each other and the R⁸ groups are joinedto form a cyclic mono- or di-substituted 1,3-dioxane or 1,3-dioxolane,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with the proviso that at leasttwo —CH₂OR⁸ are located adjacent to each other and the R⁸ groups arejoined to form a cyclic mono- or di-substituted 1,3-dioxane or1,3-dioxolane, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with theproviso that at least two —CH₂OR⁸ are located adjacent to each other andthe R⁸ groups are joined to form a cyclic mono- or di-substituted1,3-dioxane or 1,3-dioxolane, or—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with the proviso that atleast two —CH₂OR⁸ are located adjacent to each other and the R⁸ groupsare joined to form a cyclic mono- or di-substituted 1,3-dioxane or1,3-dioxolane; each R⁶ is, independently, —R⁵, —R⁷, —OR⁸, —N(R⁷)₂,—(CH₂)_(m)—OR⁸, —O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰,—O—(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷, —(CH₂) —NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

wherein when two R⁶ are —OR¹¹ and are located adjacent to each other ona phenyl ring, the alkyl moieties of the two R⁶ may be bonded togetherto form a methylenedioxy group, and wherein when two —CH₂OR⁸ groups arelocated 1,2- or 1,3- with respect to each other the R⁸ groups may bejoined to form a cyclic mono- or di-substituted 1,3-dioxane or1,3-dioxolane; each R⁷ is, independently, hydrogen or lower alkyl; eachR⁸ is, independently, hydrogen, lower alkyl, —C(═O)—R¹¹, glucuronide,2-tetrahydropyranyl, or

each R⁹ is, independently, —CO₂R⁷, —CON(R⁷)₂, —SO₂CH₃, or —C(═O)R⁷; eachR¹⁰ is, independently, —H, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷, or—CH₂—(CHOH)_(n), —CH₂OH; each Z is, independently, CHOH, C(═O),CHNR⁷R¹⁰, C═NR¹⁰, or NR¹⁰; each R¹¹ is, independently, lower alkyl; eachR¹² is independently, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷, or—CH₂—(CHOH)_(n)—CH₂OH; each Het is independently, —NR⁷, —NR¹⁰, —S—,—SO—, or —SO₂—; each g is, independently, an integer from 1 to 6; each mis, independently, an integer from 1 to 7; each n is, independently, aninteger from 0 to 7; each Q is, independently, C—R⁵ or C—R⁶; or apharmaceutically acceptable salt thereof, and inclusive of allenantiomers, diastereomers, and racemic mixtures thereof.
 2. A methodcomprising topically administering to a mucosal surface of a subject apyrazinoylguanidine compound represented by formula (I):

where X is hydrogen, halogen, trifluoromethyl, lower alkyl,unsubstituted or substituted phenyl, lower alkyl-thio, phenyl-loweralkyl-thio, lower alkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl; Y ishydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio, halogen,lower alkyl, unsubstituted or substituted mononuclear aryl, or —N(R²)₂;R¹ is hydrogen or lower alkyl; each R² is, independently, —R⁷,—(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-Z_(g)-R⁷, —CH₂)_(m)NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

wherein when two. —CH₂OR⁸ groups are located 1,2- or 1,3- with respectto each other the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane; R³ and R⁴ are each,independently, hydrogen, a group represented by formula (A), loweralkyl, hydroxy lower alkyl, phenyl, phenyl-lower alkyl,(halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, with the proviso that at least one of R³ and R⁴ is a grouprepresented by formula (A):

wherein each R^(L) is, independently, —R⁷, —(CH₂)_(n)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

wherein when two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect toeach other the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane; each o is, independently,an integer from 0 to 10; each p is an integer from 0 to 10; with theproviso that the sum of o and p in each contiguous chain is from 1 to10; each x is, independently, O, NR¹⁰, C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰,or represents a single bond; each R⁵ is, independently,—(CH₂)_(n)—NR¹²R¹², —O—(CH₂)_(m)—NR¹²R¹², —O—(CH₂)_(n)—NR¹²R¹²,—O—(CH₂)_(m)-(Z)_(g)-R¹², —(CH₂)_(n)NR¹¹R¹¹, —O—(CH₂)_(m)NR¹¹R¹¹,—(CH₂)_(n)—N^(⊕)—(R¹¹)₃, —O—(CH₂)_(m)—N^(⊕)—(R¹¹)₃,—(CH₂)_(n)-(Z)_(g)-(CH₂)_(m)—NR¹⁰R¹⁰,—O—(CH₂)_(m)-(Z)_(g)-(CH₂)_(m)—NR¹⁰R¹⁰, —(CH₂CH₂O)_(m)—CH₂CH₂NR¹²R¹²,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR¹²R¹², —(CH₂)_(n)—(C═O)NR¹²R¹²,—O—(CH₂)_(m)—(C═O)NR¹²R¹², —O—(CH₂)_(m)—(CHOR⁸)_(m)CH₂NR¹⁰-(Z)_(g)-R¹⁰,—(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹⁰-(Z)_(g)-R¹⁰,—(CH₂)_(n)NR¹⁰—O(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹⁰-(Z)_(g)-R¹⁰,—O(CH₂)_(m)—NR¹⁰—(CH₂)_(m)—(CHOR⁸)_(n)CH₂NR¹⁰-(Z)_(g)-R¹⁰,-(Het)-(CH₂)_(m)—OR⁸, -(Het)-(CH₂)_(m)—NR⁷R¹⁰,-(Het)-(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, -Het)-(CH₂CH₂O)_(m)—R⁸,-(Het)-(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, -(Het)-(CH₂)_(m)—C(═O)NR⁷R¹⁰,-(Het)-(CH₂)_(m)-(Z)_(g)-R⁷,-(Het)-CH₂)_(m)——NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,-(Het)-(CH₂)_(m)—CO₂R⁷, -(Het)-(CH₂)_(m)—NR¹²R¹²,-(Het)-(CH₂)_(n)—NR¹²R¹², -(Het)-(CH₂)_(m)-(Z)_(g)-R¹²,-(Het)-(CH₂)_(m)NR¹¹R¹¹, -(Het)-(CH₂)_(m)—N^(⊕)—(R¹¹)₃,-(Het)-(CH₂)_(m)-(Z)_(g)-(CH₂)_(m)—NR¹⁰R¹⁰,-(Het)-(CH₂CH₂O)_(m)—CH₂CH₂NR¹²R¹², -(Het)-(CH₂)_(m)—(C═O)NR¹²R¹²,-(Het)-(CH₂)_(m)—(CHOR⁸)_(m)CH₂NR¹⁰-(Z)_(g)-R¹⁰,-(Het)-(CH₂)_(m)—NR¹⁰—(CH₂)_(m)—(CHOR⁸)_(n)CH₂NR¹⁰-(Z)_(g)-R¹⁰, whereinwhen two —CH₂OR⁸ groups are located 1,2- or 1,3- with respect to eachother the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with the proviso that at least two—CH₂OR⁸ are located adjacent to each other and the R⁸ groups are joinedto form a cyclic mono- or di-substituted 1,3-dioxane or 1,3-dioxolane,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with the proviso that at leasttwo —CH₂OR⁸ are located adjacent to each other and the R⁸ groups arejoined to form a cyclic mono- or di-substituted 1,3-dioxane or1,3-dioxolane, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with theproviso that at least two —CH₂OR⁸ are located adjacent to each other andthe R⁸ groups are joined to form a cyclic mono- or di-substituted1,3-dioxane or 1,3-dioxolane; or—O—(CH₂)_(m)—NR¹⁰CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, with the proviso that atleast two —CH₂OR⁸ are located adjacent to each other and the R⁸ groupsare joined to form a cyclic mono- or di-substituted 1,3-dioxane or1,3-dioxolane; each R⁶ is, independently, —R⁵, —R⁷, —OR⁸, —N(R⁷)₂,—(CH₂)_(m)—OR⁸, —O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰,—O—(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

wherein when two R⁶ are —OR¹¹ and are located adjacent to each other ona phenyl ring, the alkyl moieties of the two R⁶ maybe bonded together toform a methylenedioxy group, and wherein when two —CH₂OR⁸ groups arelocated 1,2- or 1,3- with respect to each other the R⁸ groups may bejoined to form a cyclic mono- or di-substituted 1,3-dioxane or1,3-dioxolane; each R⁷ is, independently, hydrogen or lower alkyl; eachR⁸ is, independently, hydrogen, lower alkyl, —C(═O)—R¹¹, glucuronide,2-tetrahydropyranyl, or

each R⁹ is, independently, —CO₂R⁷, —CON(R⁷)₂, —SO₂CH₃, or —C(═O)R⁷; eachR¹⁰ is, independently, —H, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷, or—CH₂—(CHOH)_(n)—CH₂OH; each Z is, independently, CHOH, C(═O), CHNR⁷R¹⁰,C═NR¹⁰, or NR¹⁰; each R¹¹ is, independently, lower alkyl; each R¹² isindependently, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷, or—CH₂—(CHOH)_(n)—CH₂OH; each Het is independently, —NR⁷, —NR¹⁰, —S—,—SO—, or —SO₂—; each g is, independently, an integer from 1 to 6; each mis, independently, an integer from 1 to 7; each n is, independently, aninteger from 0 to 7; each Q is, independently, C—R⁵ or C—R⁶; or apharmaceutically acceptable salt thereof and inclusive of allenantiomers, diastereomers, and racemic mixtures thereof whereby thehydration of said mucosal surface is increased.